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Carbapenemase-producing Klebsiella pneumoniae represents a major public health issue globally. Isolates with resistance to the newest drugs, like ceftazidime/avibactam (CZA), are increasingly reported. In this study, we analyzed the evolution of KPC-3-producing sequence type (ST) 512 K . pneumoniae strains isolated at three different times (hospitalization days 45, 56, and 78) from the same patient, two of which were observed in a pericholecystic liver abscess. The three K. pneumoniae isolates (295Kp, 304Kp, and hmv-318Kp) from the same patient were subjected to antimicrobial susceptibility testing, whole-genome sequencing, sedimentation assay, biofilm measurement, serum resistance assay, macrophage phagocytosis, and adhesion assays. KPC-producing isolate hmv-318Kp exhibited carbapenem susceptibility, hypermucoviscous (hmv) colony phenotype and CZA resistance. Virulence markers of hypervirulent Klebsiella were absent. Two non-synonymous mutations were identified in the hmv-318Kp genome comparing with isogenic strains: a single-nucleotide polymorphism (SNP) occurred in the pKpQIL plasmid, changing bla KPC-3 in the bla KPC-31 gene variant, conferring CZA resistance; and a second SNP occurred in the wzc gene of the capsular biosynthesis cluster, encoding a tyrosine kinase, resulting in the F557S Wzc protein mutation. The Klebsiella pneumoniae strain exhibiting an hmv phenotype (hmv-Kp) phenotype has been previously associated with amino acid substitutions occurring in the Wzc tyrosin kinase protein. We observed in vivo evolution of the ST512 strain to CZA resistance and acquisition of hypermucoviscosity. The pathogenetic role of the detected Wzc substitution is not fully elucidated, but other Wzc mutations were previously reported in hmv K. pneumoniae . Wzc mutants may be more frequent than expected and an underreported cause of hypermucoviscosity in K. pneumoniae clinical isolates., Importance: Here we describe the evolution of KPC-3-producing ST512 K. pneumoniae isolated at three different times from the same patient of which the last one, from a biliary abscess, showed CZA resistance by KPC-31 production and manifested hmv colony phenotype. Hypervirulent Klebsiella pneumoniae (hv-Kp) isolates are increasingly reported worldwide. Their hypervirulent traits are associated with the presence of rmpA/A2 genes and an hmv. In this study, we identified an hmv-Kp that lacked the rmpA-D cluster but showed an amino acid substitution in the Wzc tyrosin kinase protein, involved in the capsular biosynthesis. This hmv-Kp strain emerged in vivo and evolved resistance to ceftazidime/avibactam resistance in a liver abscess of a patient. Our findings suggest that wzc mutations may be underreported, making it challenging to distinguish hv-Kp from "classic" K. pneumoniae with an hmv phenotype., Competing Interests: The authors declare no conflict of interest.

In February 2022, a critically ill patient colonized with a carbapenem-resistant K. pneumoniae producing KPC-3 and VIM-1 carbapenemases was hospitalized for SARS-CoV-2 in the intensive care unit of Policlinico Umberto I hospital in Rome, Italy. During 95 days of hospitalization, ceftazidime/avibactam, meropenem/vaborbactam, and cefiderocol were administered consecutively to treat 3 respiratory tract infections sustained by different bacterial agents. Those therapies altered the resistome of K. pneumoniae sequence type 512 colonizing or infecting the patient during the hospitalization period. In vivo evolution of the K. pneumoniae sequence type 512 resistome occurred through plasmid loss, outer membrane porin alteration, and a nonsense mutation in the cirA siderophore gene, resulting in high levels of cefiderocol resistance. Cross-selection can occur between K. pneumoniae and treatments prescribed for other infective agents. K. pneumoniae can stably colonize a patient, and antimicrobial-selective pressure can promote progressive K. pneumoniae resistome evolution, indicating a substantial public health threat.